Although western blotting is a common technique, there are still many issues that arise from the transferring step. These include the introduction of air bubbles when placing gels on membranes, and as gels have become thinner to reduce the amount of protein needed, the tearing of gels the occurs when the user moves the gels from the precast setting to the blotting membrane. These complications can be devastating when the analysis of limited amounts of protein is required, especially when no more protein is available or it is a clinical specimen. Furthermore, the concept of a protein separation and transfer combination unit is hindered by the current use of insulating plastics to house precast gels. Additionally, researchers often prefer to watch protein separation during electrophoresis, which limits the use of non-transparent materials as the gel supporting structural plates.
Simplifying the western blotting technique reducing the traditional two step process (protein separation and protein transfer) into a single step and using a single apparatus instead of separate apparatuses for electrophoresis and for protein transfer. However, one challenge in creating a single combination gel electrophoresis and protein transfer unit involves creating an apparatus where the user can visualize the degree of protein separation during electrophoresis and thereafter transfer the proteins to a blotting membrane without physically transferring the gel to the membrane. For such an apparatus to perform both electrophoresis and protein transfer, the plates must form the structural support for the gel, but also be able to transfer current through the gel supporting plates to the blotting membrane. The challenge is that the electrical current required to transfer proteins to the blotting membrane must run perpendicularly to the current required to separate proteins during electrophoresis.
There have been many attempts to simplify the separation and transfer of proteins and other macromolecules using various apparatuses and techniques. U.S. Pat. No. 4,994,166 to Fernwood et al. describes a single apparatus for slab gel electrophoresis and blotting, both of which are performed in a single tank cell having separation electrodes along opposing vertical walls, and blotting electrodes arranged horizontally above and below the level of the gel. The cell is operated in separatory and blotting modes, in which separatory and blotting electrodes are separately energized. Fernwood requires porous gel supports to allow the electric field to pass through the membrane and the top plate transfer electrode must be removed from contact with the buffer solution during the separatory phase. Fernwood also discusses some of the problems of using gel-supporting plates for both electrophoresis and transfer of proteins. Since there must be a potential difference sustained between the two ends of the gel during separation phase, conductive blotting electrodes in contact with the buffer solution during separation nullify the field around the gel, thereby preventing protein migration and separation. Fernwood solves this problem by using a wire array as the lower blotting electrode, and in order to avoid the nullifying field, the electrode must either be raised above or lowered below the liquid buffer level in the tank. Another solution offered is that the height of the electrode plate may be fixed, and the buffer level raised and lowered as necessary to establish or break electrical contact with the gel supporting plate. Raising or lowering the plate or buffer between electrophoresis and transfer phases requires an additional level of complexity to the apparatuses and methods for separating transferring proteins to a blotting membrane.
U.S. Pat. No. 5,102,524 to Dutertre describes an electrophoresis device and method to control migration of macromolecules through gel plates, where different sets of electrodes are used in a two-step process to first separate macromolecules (e.g. proteins) and then to transfer the molecules to a blotting membrane.
U.S. Pat. No. 5,593,561 to Cognard also describes an electrophoresis device and method for controlled migration of macromolecules and transfer thereof to a membrane in a vessel. The first electric field, established between electrodes, provides means for macromolecular separation in a gel, and the second electric field, perpendicular to the first, provides means for transferring the macromolecules onto the membrane. In the described method, electrodes and blotting membranes are assembled in the vessel, which is then filled with gel. The gel is then liquefied, allowing the removal of the membrane. Cognard's device is for use without a prefabricated gel and membrane unit.
U.S. Pat. No. 8,173,002 to Margalit discloses a dry blotting system to transfer proteins onto a blotting membrane. The system does not include an electrophoresis device, so the device does not allow the user to visualize separation and blotting of proteins in a single device. The device requires the user to be present to transfer the gel to a blotting membrane on the blotting device. Margalit teaches the use of electrically conducting polymers, but not in combination with a single device that can both separate proteins and transfer the proteins to a blotting membrane. Margalit does not teach the use of transparent gel supporting plates so that the user can visualize protein separation during electrophoresis.
U.S. Patent Appl. Pub. No. 2006/0042951 to Ohse discloses an apparatus to separate and transfer proteins via the use of a fine grove, a transferring electrode and a transparent conductive material having a thickness of approximately 0.1 μm. The apparatus includes a pair of separating electrodes for causing a substance in a sample to move along a passage, and a pair of transferring electrodes for causing the substance in the sample to be transferred to the capturing material by electrophoresis. The transparent conductive material is not capable of being the support structure due to its thickness of approximately 0.1 μm, which would not have sufficient rigidity to serve as the supporting walls for a gel. The separation and blotting is performed in an electrophoresis buffer and does not make use of a gel slab or gel slab assembly, which are commonly used for western blots.
U.S. Pat. No. 6,602,391 to Serikov discloses an apparatus and method for capillary separation of macromolecules and post-separation blotting. However, Serikov does not disclose the use of a slab gel where the user can view the separation of macromolecules and transfer the macromolecule to a blotting membrane for western blotting.
Conductive polymers have previously been described, but not in conjunction with electrophoresis and blotting. Ates et al. describes numerous applications of conducting polymers in “Conducting Polymers and their Applications” (Current Physical Chemistry, 2012, 2, 224-240). International Patent Application No. PCT/EP2013/065163 to Jung discloses a conductive polymer composition and transparent electrode for an antistatic layer. International Patent Application No. PCT/KR2008/002236 to Kim discloses a conductive polymer for use as a transparent electrode and the method of fabricating the electrode using an ink jet spray method. U.S. patent application Ser. No. 13/616,804 discloses a transparent panel and method of manufacturing a transparent panel where a conductive polymer layer is formed to make a transparent electrode.
Transparent conductive plates using conductive polymers have not been used in electrophoresis and blotting apparatuses where the conductive plates are used as both the gel supporting structures so that the pre-cast gel and its conductive polymer housing can be used for both electrophoresis and blotting without removing the gel after protein separation and still be used as the gel supporting structure as the proteins are transferred to the blotting membrane.
There currently remains a need for devices and associated methods that can perform gel electrophoresis and protein transfer in a single precast gel and membrane combination unit. Additionally, since researchers prefer to watch the electrophoresis steps during the electrophoresis phase, there remains a need develop gel structural supporting plates that allow visualization, but can also be used in both the electrophoresis phase and protein transfer phase of a western blot.
All patents, patent applications, and non-patent applications disclosed in the background and description of the invention are hereby incorporated by reference for all purposes in their entireties.